Fluorescent nanoscopy method

ABSTRACT

An analysis of an object dyed with fluorescent coloring agents carried out with the aid of a fluorescent microscope which is modified for improved resolving power and called a nanoscope. The method is carried out with a microscope having an optical system for visualizing and projecting a sample image to a video camera which records and digitizes images of individual fluorescence molecules and nanoparticles at a low noise, a computer for recording and processing images, a sample holder arranged in front of an object lens, a fluorescent radiation exciting source and a set of replaceable suppression filters for separating the sample fluorescent light. Separately fluorescing visible molecules and nanoparticles are periodically formed in different object parts, the laser produces the oscillation thereof which is sufficient for recording the non-overlapping images of the molecules and nanoparticles and for decoloring already recorded fluorescent molecules, wherein tens of thousands of pictures of recorded individual molecule and nanoparticle images, in the form of stains having a diameter on the order of a fluorescent light wavelength multiplied by a microscope amplification, are processed by a computer for searching the coordinates of the stain centers and building the object image according to millions of calculated stain center co-ordinates corresponding to the co-ordinates of the individual fluorescent molecules and nanoparticles. With this invention it is possible to obtain a two-dimensional and a three-dimensional image with a resolving power better than 20 nm and to record a color image by dyeing proteins, nucleic acids and lipids with different coloring agents.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to equipment for scientific research, and lensfluorescence microscopes in particular, that are used for obtainingimages of fluorescent objects immobilized on a glass. This inventionalso relates to a computerized fluorescent-microscopically method ofreconstructing images of objects with resolution up to severalnanometers (nm).

2. Discussion of Related Art

There are known optical microscopes which can create zoomed images of anobject with object lenses that can show two spots on an objectseparately only when the distance between them is more than so calleddiffraction limit. This limit can be calculated using following formula:r=0.61λ/A (1), where λ is light wavelength for light collected by objectlens with aperture A=n*sin(φ), n is a refraction index of substancewhich surrounds object spots, φ is an angle between object lens axis andextreme rays which fall into object lens and are detected in detector.Now, different types of devices are used for fluorescent microscopy byobject lenses. Powerful arc-lamps, incandescent lamps, laser or sunlight can be light sources for the microscope. Fluorescence starts inall dye molecules are present in a lighted area. The area is lightedthrough the object lens using a light-dividing dichroic mirror that letsexiting light to fall on the object and reflects fluorescence light tothe detector. The second type of lighting occurs by sending laser lightfrom the side and lights the object all the way down or through anobject glass at a total internal reflection angle. In this case, lightreaches a depth of only 0.3 of light wavelength from border betweenglass and an object which has a refraction index lower than glass.Object fluorescent light is collected by an object lens and sends anobject image for visual observation and registration by aphotomultiplier, a photographic tape or a digital video-camera. The maindisadvantage of all existing lens microscopes is that they have a limitof resolution for two neighboring spots and the limit can be calculatedaccording to the above formula (1).

Recently, microscopy using super lenses made from silver film has beendeveloped. A film thickness less than 50 nm can assure resolution of twospots on a distance of approximately 50 nm from each other. (N. Fang andX. Zhang, Imaging properties of a metamaterial superlens, 2003, App physLet v. 82, 2, 161-163; Nicholas Fang, Zhaowei Liu, Ta-Jen Yen, and XiangZhang Regenerating evanescent waves from a silver superlens, 2003,OPTICS EXPRESS, VoI. 11, No. 7, 682-687). Use of such microscopes onbiological objects is unclear. The present microscope model has aresolution several times lower than a resolution of the device that wepropose.

There are devices with a maximum resolution better than 1 nm, forexample, electronic, tunnel, and atomic-force microscopes that have notonly real advantages but also serious disadvantages, such as: complexityand expensiveness of their design and work with objects; lack ofopportunity to receive a color image for distinguishing molecules ofdifferent types; objects that usually should be dried and treated withsubstances which change the mutual layout of different parts of theobject. Atomic and tunnel microscopes also do not detect structuresinside the object; only one spot can be detected at a time and ascanning speed does not overcome 1 square micron per min; and an end ofthe needle easily becomes dirty and does not then reach an objectsurface.

There is also a device, where object fluorescence is exited by laserthrough a pinhole on the tip of a glass fiber. The fiber is moved bydrives in three directions to position an end of the fiber nearlight-reflecting, light-diffusing or covered with fluorescent moleculessurface. This type of microscope does not use lenses and permitsobtaining images with resolution ten times better than the resolution ofcommon optical microscopes. These results can be reached only when thepinhole on the tip of glass fiber is much less in diameter than in lightwavelength. The light comes onto an object with a depth much shorterthan a light wavelength. Practically all light returns back into theglass fiber, except that part which was captured by objects from outsideof the hole. Fluorescence, light-diffusion, and reflected lightstrength, captured in the object near the hole, is measured by aphotomultiplier. The image of an object surface is reconstructed by acomputer, which gathers information about the strength of measured lightand data on an end of glass fiber coordinates. The main disadvantages ofthis system are: a need to use expensive high-precision and fast-actingmechanic units, responsible for moving of glass fiber against object;producing glass fiber with an end hole less than 50 nm in diameter isvery expensive, complex and difficult for duplicating; the hole iseasily dirtied and is not able to then reach an object surface; only aninsignificant part of light can leave the fiber through the hole with adiameter less than light wavelength; increases of light intensity leadsto overheating and destruction of the fiber end; it is impossible todetect fluorescence in the areas of the object which are not accessibleby glass fiber; and only one spot can be detected at a time, and surfacescanning speed does not exceed 1 square micron per minute.

One more new microscope type scans an object surface with several lightbeams simultaneously. National Institute of Standards and Technology(NIST), issued a grant for 5 years research work on creation of thismicroscope (betterhumans.com, Article ID 2005-02-11-4, “OpticalMicroscopes Enter the Nano Age. Hybrid system being developed to imageand measure features smaller than the wavelength of visible light”.) Thearticle states that a 40 nm nanoparticle can be distinguished using thismethod. There are no indications of authors being successful indistinguishing two separate particles located on a distance less than 40nm from each other. It is not clear from presented drawings andexplanations how this method will allow distinguishing two particles ondistance less than r<0.61λ/A between them and the suggested device willlikely not reach resolution of object details, located on a distancemuch shorter than light wavelength.

One method of using common fluorescent microscope (Erwen, A Sharonov, JH Ferris, R M Hochstrasser: Direct visualization of nanopatterns bysingle-molecule imaging. App Phys Let 2005, 86: 043102) can be thoughtof as relevant to this invention. The main idea of this method is that asample, a light film of polymer with free open spherical cells 1 micronin diameter, is dyed with very low concentration of fluorescent peptide.Such concentration allows to observe separate peptide molecules whichcan migrate in Brownian motion inside the hollow of spheres. The sampleis lighted through an object glass by a laser beam. A lightning angle isequal to a total internal reflection angle. A laser beam excitesfluorescence in a 150-200 micron layer of the sample near the glass. Alocation of several tens of molecules of fluorescing peptide, each ofwhich was dyed by simultaneously fluorescing molecules, was detected bya high-sensitivity video camera (Roper Scientific, Cascade 512F withelectrons multiplier built in CCD) in 500 sequential frames. Each framewas recorded to computer memory. Each image contained many spots ofapproximately 0.5 microns in diameter multiplied on a system zoom value.All these images were added to each other and a resultant image had aresolution not exceeding a resolution of a common fluorescentmicroscope. Main disadvantages of this system are: there is noinformation in the article about opportunity to calculate a location ofdetected spot centers and to generate an image on the basis of thesespots with resolution higher than a diffraction limit (see formula 1);approaches to selective dyeing of object structures are not described inthe article; and there is no description of solving that after dyeing,substances lose color in the process of detecting non fluorescingpeptide molecules staying in an observation area. This will make thesolution more thick and will not allow replacing such molecules with newfluorescing ones. This does not allow obtaining larger quantity offrames than 500. These large quantities are needed to receive image withresolution higher than allowed by a diffraction limit (see formula 1).

SUMMARY OF THE INVENTION

Researching of biological objects, for example muscles, includes thefact that many different types of molecules are located much closer thanthe resolution of common optical microscopes which create a zoomed imageof the object by an object lens in a plane of a recorder, eye, photo orvideo camera. The diffraction limit of microscope resolution (formula 1)limits the resolution in microscopes with simultaneous observation ofall spots in the observation area and in microscopes with sequentialobservation of all spots of an object by a light ray being focused onone spot (confocal and other types of scanning lens microscopes). Thatis why it is good if all the advantages of optical microscopes and usedmethods of selective dyeing of different types of molecules in a visionarea are united with such improvement of resolution, which would allowseparate observation of molecules located less than 10-20 nm from eachother. This invention provides methods of dyeing objects, preparingobjects for research, computer analysis of results. This procedure willallow receiving an object image with resolution higher than 20 nm. Thiswill turn a fluorescent microscope into a “nanoscope”. This task issolved by making multiple pictures of low dyed objects, (all fluorescingmolecules in the objects are seen separately as spots with a diameter of2r=1.22λ/A, having different location on tens of thousands of sequentialframes). Further, all these frames are used to calculate locations ofcenters of all detected spots (the locations correspond with actualcoordinates of fluorescing molecules). Then 3-D reconstruction of alldye molecule locations is performed. Resolution is comparable with afluorescing molecule size. Different structures of the object can bedyed in different colors. Fluorescent nanoscopes can be based onstandard modules, used in fluorescent microscopy. This inventioncomprises various modules: an optical system for visual observation andtransmitting of object image to digital video camera. Video camerasshould be able to detect and digitize images of separate fluorescingmolecules and nanoparticles with low background noise level. Secondmodules of the system is computer for recording and analyzing images. Athird component is a sample holder, located opposite to objective lens.A fourth component is a set of changeable suppressing color filters forpicking up light of sample fluorescence. The nanoscope should beequipped with two light sources installed aside from a sample holder. Aninstallation angle should assure lighting of an entire slice of thesample on full depth or in a layer less than 150 nm near the glass.Fluorescent molecules in this layer can absorb energy of light waves,flowing through the border when lighted on a total internal reflectionangle. Observation objects should be preliminary dyed in a solution witha saturating concentration of caged dye which starts fluorescing onlywhen a UV light pulse separates blocking fluorescence groups from dyemolecules. Superfluous dye should be carefully washed out. It is veryhard to assure stability of molecular structures for a long time farfrom covering a glass and a prism glass holding the object. This is truefor objects observed exactly in a fresh flushing solution. That is whythe best resolution of a nanoscope can be received only for those layersof the object which are located on the distance not more than 150 nmfrom the prism glass. This is the depth which light reaches when fallingon the boundary surface at a total internal reflection angle. In case ofobserving a dead preserved object it is usually possible to observe theobject during many hours. Different structures, located near a baseglass are almost an absolute immovable. They keep their location becauseof multiple stable mutual connections and connections with the baseglass. They are immovable in spite of the fact that nearly 80% of volumeis occupied with an aqueous solutions of different salts. Gaps on glassedges should be hermetically sealed (for example, with paraffin) toavoid drying of the solution.

3-D nanoscopy requires total immobility of the object and its partsagainst an objective lens. That is why the object, after being dyed withcaged dye and flushed, should be treated with dehydrating solutions andplaced into a polymer non-fluorescent substance (for example, epoxy).Microscopic sections of the object of several microns thickness can beused for 3-D reconstruction of the object with 10-20 nm resolution inall three dimensions. Such an object contained in a solid substance canbe lighted as it is done in common fluorescent microscopy. Only animprovement of an additional flash-lamp should be used in order to turnseveral hundred non-fluorescing molecules into fluorescing in eachframe. For both, object in liquid and object in polymer film, a UVflash-lamp with λ<360 nm periodically lights the object, dyed withnon-fluorescing dye, and each time turns several hundred (and even athousand) of non-fluorescing molecules into fluorescing ones byphotolysis of special chemical groups blocking fluorescence. The laserconstantly lights the object and excites fluorescence of newly formedfluorescent molecules with such strength, that each of them will sendseveral tens of thousands of light quanta in part of a second. Moleculeswill photobleach after registration of its fluorescence on a digitizedframe with low background noise level. Cycling of fluorescent moleculesgeneration, their excitation, registration and photobleaching can berepeated tens of thousands of times. Each time new fluorescencemolecules will be generated, detected and photobleached. Not convertednon-fluorescent molecules will not photobleach because they do notabsorb laser light. Tens of thousands of frames can be detected usingthis method. Hundreds and even thousands of fluorescing molecules can bedetected on each frame. It will allow calculation of locations of alltens of millions of fluorescing molecules covering surfaces of allstructures, located in a field of vision. Dyes with different activegroups can be used for dyeing structures of different nature indifferent colors. Active groups can link to either proteins, eithernucleic acids, either fats, etc. (one suggested method name is colornanoscopy).

The second modification of a nanoscopy method is based on the fact thatthe Brownian motion of very bright fluorescing andphotobleaching-resistant nanoparticles is possible inside most parts ofvolume of a biological sample with penetrated membranes, filled withsalt solution. Nanoparticles are presented by ficobiliprotein moleculesor fluorescing micro spheres 10-40 nm in diameter. This motion can beregulated by electrophoresis with current, provided by several pairs ofinterlacing electrodes. Current directs the particles motion indifferent directions, so they scan all accessible volume. If severalhundreds of the same, or changing with those which are out of lightedarea, fluorescing particles that are moving on the distance more than1-2 microns from each other are detected in each frame, then it ispossible to calculate their location with several nm accuracy, ascoordinates of centers of corresponding spots. Coordinates from allframes can be used for finding out all locations where particles canappear over a long time period. It indicates all volume that is occupiedwith liquid. Part of the volume, where particles were not able to bepresent, can be considered to be occupied with dense structures ofdifferent origin, peptides, chromosomes, parts of not damaged membranes,etc. Possible fluctuations of a particle location can enlarge a spotsize a little, but a spot center location can be considered as anaveraged coordinate of the particle in the object. It should be noted,that particles with positive charge, negative charge, and neutralparticles can visit different areas of the object because they interactwith local charges of bio-structures, and that is why this method can beuseful for studying surface charges of object structures, such as asuggested name of the method being monochrome nanoscopy.

Both suggested methods can assure a nanoscope resolution up to severalnm, depending not from resolving power (formula 1), but from intrinsicmobility of structures in an observed part of the object during a longtime period. A second factor is a proportion between noise and signal invideo data. Both factors can be adjusted by various modifications ofpresented methods. For example, some bright shining key fluorescingparticles can be introduced into the object. Then coordinates ofchanging particles can be calculated taking into account moving in avision area of key particles rigidly connected with the object. Suchapproach allows reaching 2-D distinguishing parts of the object, locatedon the distance of several nm.

It should be noted that some parameters of fluorescing particles images,a diameter of spots and light intensity distribution along a section ofeach spot, can change as particle distances from a focal plane of amicroscope object lens. That is why it is suggested to perform acalculation of these parameters in order to calculate a third coordinateof the shining particle. It is suggested to project an object image ontwo video-cameras. This will improve precision of the third coordinatedefinition. The light rays are divided in two rays after the objectlens. Video-cameras are located so that two neighboring focal planes ofthe object would be projected on them through one object lens. Lightintensity distribution for each particle will be different when detectedby different cameras. It can be scaled depending from the thirdcoordinate and used to create 3-D images of the objects. FIG. 3 shows anillustration of 3-D nanoscopy foundations. A diameter and intensity ofspots from fluorescing particles which are projected on CCD, dependsfrom a distance between the particle and the object lens. Two cameras,focused on different focal planes, show one particle with differentdiameters and a different intensity distribution along diameters. Thediagrams in the upper right corner of the figure illustrate differencesbetween diameters and a distribution of intensity along diametersdepending from cameras and a fluorescing particles location.

A nanoscope in some cases can be helpful in finding centers of spotswhich are created by fluorescing particles in two-three differentwavelengths and same exciting status. It can be done by separating lightdichroic mirrors that pass light with one wavelength to one camera andreflect light with the other wavelength to the other camera. Thus, thisinvention allows to reconstruct simultaneously with a nano resolutionlocation of several different types of object molecules. These moleculeswill be dyed with dyes having different emitting wavelengths which willallow enlarged quantities of simultaneously seen and detected separatelyspots from fluorescing particles. This can be achieved because they canbe seen separately in waves of different lengths, even if correspondingspots of different colors are seen interfering.

It can be very useful if described methods of nanoscope work arecombined with an additional changing of caged non-fluorescing moleculesinto fluorescing ones. This can be done by chemical reactions ordifferent physical influences which will separate groups which blockfluorescence. Examples of such reactions are ATP reactions, esteraseinfluence, peroxidation, radioactive transformation, etc. Fluorescingmolecules, created as result of such modifications, can be exited bylaser and detected with video-cameras, used for calculating coordinatesof an object structure surface according to above described methods.Their coordinates can be calculated as centers of appropriate spots onvideo frames. Then their images can be “overlapped” on an earlierreconstructed image of the object. They will show a location of reactionactive groups of the object. Resolution of such image will be betterthan 20 nm.

This invention is described in the attached claims, and can be modifiedwith different methods, not stepping aside from basic ideas of thisinvention.

Operation of the device in nanoscope mode can be altered by using afluorescence microscope in the normal mode. This mode gives opportunityof direct visual observation of the object in situ with commonresolution (formula 1). It also gives an opportunity to detect an imagewith a digital video-camera of very high sensitivity and to transmit itto computer memory. Then these frames will be analyzed to measuregeometric parameters and light intensity in different areas of theframe. This can be used, for example, during bioluminescent andchemoluminescent research.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings show how a fluorescent microscope can be modified toimplement a nanoscopy method according to this invention.

DETAILED DESCRIPTION OF THE INVENTION

The fluorescent microscope-nanoscope, as it is shown on FIGS. 1 and 2,is equipped with: one (FIG. 1) and two (FIG. 2) monochrome video-cameras(1) with digital output and suppressing color-filters, located oppositeto their sensors (CCD). These color-filters pass only fluorescent lightto cameras. A microscope is also equipped with a light dividingremovable prism (2); object lens (3) with zoom up to 100× and anaperture up to A=1.4. Object (4) is pressed down to glass object holder(5) which is beveled in the form of truncated prism edges. The devicealso has laser (6) with lens system for exciting fluorescence throughprism planes; with a pulse UV-source with a lens system for photolysisof blocking fluorescence groups, present on dye molecules. Anotherimportant part is computer (8) with software for recording and workingwith digitized images. This software is also used for control of a powersource, and providing energy for UV pulses and electrophoresis device.Eye lens (10) (figure 1) is used for visual observation (9). Unit shouldbe placed into soundproof cabinet, placed on an anti-vibration table.The device, providing power to electrophoresis electrodes which directmovement of mobile fluorescing nanoparticles inside the object, filledwith salt solution, will be described separately.

This is an example of receiving an image with a nanoscope which useshigh sensitivity cameras Cascade 1K, such as produced by Photometrics,or cameras SIS1_t285EM, or produced by Theta-system. SIS1_t285EM camerasare equipped with CCD TC285SPD, produced by Texas Instruments, withelectrons multiplier which is included in CCD crystal. It has a quantumeffectiveness up to 63%, 1004 active horizontal square pixels with sideof 8 microns in 1002 lines on a photosensitive square of approximately8×8 mm. If an object image is projected directly to CCD of avideo-camera by a 100× object lens, then square on the object, lightedwith a laser and its lens system, should be a little bit larger than80×80 microns. Each CCD pixel corresponds with an object square of 80×80nm. Each shining particle or molecule is seen on the object as a spotwith a diameter of 2r=1.22λ/A, which is near 560 nm. It will beprojected to a CCD square with a diameter near 7 pixels. The averagedistance between chaotically spread particles in each frame should bemore than 2000 nm. In this case, practically all of them will be seenseparately. In this case, at least 40×40=1600 spots can besimultaneously projected to CCD in each frame in such a manner, thatthey will be observed separately. Different methods can be used to reacha concentration of simultaneously fluorescing particles. First, it ispossible to dye an object with brightly fluorescing particles in lowconcentration. This will allow them to move in an observation area in aBrownian motion and they can be additionally moved with anelectrophoresis current. Current is provided with several pairs ofelectrodes in such manner that it will direct particles to move indifferent directions. Secondly, the object can be dyed with special dyeslike 5-carboxyfluorescein-bis-(5-carboxymetoxy-2-nitrobenzil) ester,beta-alanin-carboxylamid, suxynimidil ester (CMNB-cagedcarboxyfluorescein, SE), fabricated by Molecular Probes, USA. Afterlighting with a UV flash with a wavelength of 310-365 nm (with apreviously adjusted exposition) approximately 1000-1600 molecules ofthis non-fluorescing dye in an observation area will loose specialgroups which block fluorescence. Such molecules are able to produceseveral thousands quanta of green light when lighted with blue light.After this they will photobleach. When an objective lens aperture isA=1.1-1.3, more than 10% of light from each fluorescing particle will betransmitted to a video-camera and participate in formation of the spot,covering near 40 pixels of CDD. Light intensity in the center of thespot is up to 100 quanta. This is enough to receive a video signal withquite a satisfactory proportion of signal and noise (when using theabove mentioned cameras). This proportion will allow calculatingcoordinates of a spot center with resolution higher than 20 nm. This dyedoes not absorb laser light and photobleach while groups which blockfluorescence are linked to it. That allows repeating the followingprocess tens of thousands of times: in the beginning CCD detects theimage of the object with residue fluorescence. Then the image isdigitized in a video-camera and is transmitted to computer. Then UVflash creates 1000-1600 fluorescing molecules in a vision area, andlaser excites fluorescence in these molecules. CCD detects images offluorescing molecules and particles, overlapped on residualfluorescence. The image is digitized in the video-camera and istransmitted to a computer where previous frame with a residuefluorescence is subtracted from it. The received subtracted frame isstored in computer memory for further analysis with the purpose ofcalculating coordinates of a spot center, its averaged diameter, andintensity. Then the object is for some time lighted with laser withoutdetecting its image to maximize photobleaching of already detectedmolecules. Then the whole cycle is repeated. New fluorescing moleculesare created, detected and photobleached in each cycle until coordinatesof all dye molecules are detected. Molecular Probes produce not onlymentioned dye. Dyes can be produced on request. It also produces sets ofreagents for individual creating of such dyes, such as a “caging kit”(D-2516).

Cameras, indicated in the example, allow recording images of separatefluorescing molecules with a frequency of 10 frames per second. Imagesare recorded with satisfactory proportion of signal and noise. It ispossible to detect, for example, 40000 frames during several hours. Eachframe will contain up to 1600 images of molecules. If so, a totalquantity of detected fluorescing molecules' coordinates can reach 64million, an average distance between observed separately molecules canbe only 10 nm. This is tens times better than in any other lensmicroscopes. Frames with molecule images should be saved withoutcompressing or compressed without loosing quality. This should be donefor more exact calculating of molecule coordinates. A total volume ofinformation on all not compressed frames in one experiment can reachtens of gigabytes. This will not create a problem taking into accountmodern hard disk drives volume. Literature sources provide informationon various algorithms of calculating spot centers. Computer software hasbeen created to perform such calculations and reconstruction of a fullimage based on a table of calculated coordinates of spot centers.

1. A method for nanoscopy of dyed objects being placed in a solution oras a slice of a preserved sample being placed in a polymer, wherein afluorescent microscope having digital video-cameras with suppressingcolor filters for picking up fluorescence light of the object that areable to detect with a low noise level images of separate fluorescingmolecules excited by a laser, the microscope having a computer forrecording images received from the video-camera, the method comprising:periodically turning one to two thousands of non-fluorescing dyemolecules into fluorescing fluorescing dye molecules by a UV flash,wherein the molecules cover object structures, turning the moleculesinto fluorescing molecules by separating chemical groups which blockfluorescence, detecting images of created fluorescing molecules onvideo-camera frames as separate spots, wherein the molecules photobleachin the process of detection and frames with residual fluorescence aresubtracted from frames containing a mixture of fluorescing particles anda residual fluorescence, saving subtracted frames to computer memory tofind centers of spots which represent coordinates of dye molecules,repeating the process a number of times to calculate coordinates of alldye molecules in a lighted area of the object and reconstruct a 2-Dimage or a 3-D image of the object with a resolution better than 20-50nm.
 2. The method according to claim 1, wherein in lieu of fluorescingmolecules, a regularly created and discolored object is dyed withobserved separately fluorescing nanoparticles each containing a numberof fluorescing light resistant molecules that can move betweenstructures of the object because of Brownian forces and when driven by a2-D electrophoresis a laser constantly excites the fluorescence of theparticles, wherein the video-camera and the computer detect tens ofthousands of frames with one to two thousands of observed separatelyspots in each frame reflecting a location of particles, and a liquidpart of the area that is accessible to the particles is detected basedon calculated spot centers coordinates on all frames, together withcalculation of inaccessible parts of the area that is occupied byimmobile structures of an object.
 3. The method according to claim 1,wherein a second video-camera is used and is the same as the firstvideo-camera and is located so that a sensor of the second video-camerais focused on an object plane which differs in depth from a focal planeof the first video-camera from 50 to 2000 nm, a difference indistribution of light intensity received by both of the video-camerascalculates a third coordinate of a dye molecule location in an objectused for creating a 3-D image of the object, the object in liquid sampleis lighted through an object glass under an angle equal to a total angleof internal reflection, the object being solidly cut with a thicknessfrom a part of a micrometer to several micrometers can be lightedthrough an objective lens on an all depth of solid cut or through theobject glass under the total angle of internal reflection.
 4. The methodaccording to claim 1, wherein different structures of the object aresimultaneously dyed with different dyes fluorescing on differentwavelengths, and a set of dichroic mirrors and color-filters selectivelyprojects different structure images to two or more video-cameras whichmultiplies a quantity of simultaneously detected molecules.
 5. Themethod according to claim 4, wherein in addition to an image formed,different caged molecules are periodically introduced into the object,the molecules can turn a non-fluorescing molecule into a fluorescingmolecule by different chemical reactions or because of differentphysical exposures, factors detach chemical groups which blockfluorescence, the groups are detach with a speed so that all generatedfluorescing molecules are detected on video-camera frames as separatespots, molecules photobleach during registration, frames with residualfluorescence are subtracted from frames containing mixture of images ofresidual fluorescence and fluorescing molecules images, subtractiveframes are saved in computer memory for further finding of centers ofspots that represent coordinates of dye molecules, and the cycle isrepeated a number of times by creating new fluorescing molecules inlighted area to calculate coordinates of all dye molecules in the objectand to perform computer reconstruction of a location of specialmolecules on an earlier received 2-D image or 3-D image of the object.6. The method according to claim 3, wherein in addition to an imageformed, different caged molecules are periodically introduced into theobject, the molecules can turn a non-fluorescing molecule into afluorescing molecule by different chemical reactions or because ofdifferent physical exposures, factors detach chemical groups which blockfluorescence, the groups are detach with a speed so that all generatedfluorescing molecules are detected on video-camera frames as separatespots, molecules photobleach during registration, frames with residualfluorescence are subtracted from frames containing mixture of images ofresidual fluorescence and fluorescing molecules images, subtractiveframes are saved in computer memory for further finding of centers ofspots that represent coordinates of dye molecules, and the cycle isrepeated a number of times by creating new fluorescing molecules inlighted area to calculate coordinates of all dye molecules in the objectand to perform computer reconstruction of a location of specialmolecules on an earlier received 2-D image or 3-D image of the object.7. The method according to claim 2, wherein in addition to an imageformed, different caged molecules are periodically introduced into theobject, the molecules can turn a non-fluorescing molecule into afluorescing molecule by different chemical reactions or because ofdifferent physical exposures, factors detach chemical groups which blockfluorescence, the groups are detach with a speed so that all generatedfluorescing molecules are detected on video-camera frames as separatespots, molecules photobleach during registration, frames with residualfluorescence are subtracted from frames containing mixture of images ofresidual fluorescence and fluorescing molecules images, subtractiveframes are saved in computer memory for further finding of centers ofspots that represent coordinates of dye molecules, and the cycle isrepeated a number of times by creating new fluorescing molecules inlighted area to calculate coordinates of all dye molecules in the objectand to perform computer reconstruction of a location of specialmolecules on an earlier received 2-D image or 3-D image of the object.8. The method according to claim 1, wherein in addition to an imageformed, different caged molecules are periodically introduced into theobject, the molecules can turn a non-fluorescing molecule into afluorescing molecule by different chemical reactions or because ofdifferent physical exposures, factors detach chemical groups which blockfluorescence, the groups are detach with a speed so that all generatedfluorescing molecules are detected on video-camera frames as separatespots, molecules photobleach during registration, frames with residualfluorescence are subtracted from frames containing mixture of images ofresidual fluorescence and fluorescing molecules images, subtractiveframes are saved in computer memory for further finding of centers ofspots that represent coordinates of dye molecules, and the cycle isrepeated a number of times by creating new fluorescing molecules inlighted area to calculate coordinates of all dye molecules in the objectand to perform computer reconstruction of a location of specialmolecules on an earlier received 2-D image or 3-D image of the object.